Soybean promoter ltp4 and flower-preferred expression thereof in transgenic plants

ABSTRACT

The promoter of a soybean lipid transfer protein LTP4 and fragments thereof and their use in promoting the expression of one or more heterologous nucleic acid fragments in plants are described.

This application claims priority to U.S. provisional Application No.61/014,567 filed Dec. 18, 2007, the entire contents of which are herebyincorporated by reference.

FIELD OF THE INVENTION

The present invention relates to the field of plant molecular biology,more particularly to regulation of gene expression in plants.

BACKGROUND OF THE INVENTION

Recent advances in plant genetic engineering have opened new doors toengineer plants to have improved characteristics or traits, such asplant disease resistance, insect resistance, herbicidal resistance,yield improvement, improvement of the nutritional quality of the edibleportions of the plant, and enhanced stability or shelf-life of theultimate consumer product obtained from the plants. Thus, a desired gene(or genes) with the molecular function to impart different or improvedcharacteristics or qualities can be incorporated properly into theplant's genome. The newly integrated gene (or genes) coding sequence canthen be expressed in the plant cell to exhibit the desired new trait orcharacteristic. It is important that appropriate regulatory signals bepresent in proper configurations in order to obtain the expression ofthe newly inserted gene coding sequence in the plant cell. Theseregulatory signals typically include a promoter region, a 5′non-translated leader sequence and a 3′ transcriptiontermination/polyadenylation sequence.

A promoter is a non-coding genomic DNA sequence, usually upstream (5′)to the relevant coding sequence, to which RNA polymerase binds beforeinitiating transcription. This binding aligns the RNA polymerase so thattranscription will initiate at a specific transcription initiation site.The nucleotide sequence of the promoter determines the nature of the RNApolymerase binding and other related protein factors that attach to theRNA polymerase and/or promoter, and the rate of RNA synthesis.

It has been shown that certain promoters are able to direct RNAsynthesis at a higher rate than others. These are called “strongpromoters”. Certain other promoters have been shown to direct RNAsynthesis at higher levels only in particular types of cells or tissuesand are often referred to as “tissue specific promoters”, or“tissue-preferred promoters”, if the promoters direct RNA synthesispreferentially in certain tissues (RNA synthesis may occur in othertissues at reduced levels). Since patterns of expression of a chimericgene (or genes) introduced into a plant are controlled using promoters,there is an ongoing interest in the isolation of novel promoters thatare capable of controlling the expression of a chimeric gene (or genes)at certain levels in specific tissue types or at specific plantdevelopmental stages.

Among the most commonly used promoters are the nopaline synthase (NOS)promoter (Ebert et al., Proc. Natl. Acad. Sci. U.S.A. 84:5745-5749(1987)); the octapine synthase (OCS) promoter, caulimovirus promoterssuch as the cauliflower mosaic virus (CaMV) 19S promoter (Lawton et al.,Plant Mol. Biol. 9:315-324 (1987)); the CaMV 35S promoter (Odell et al.,Nature 313:810-812 (1985)), and the figwort mosaic virus 35S promoter(Sanger et al., Plant Mol. Biol. 14:433-43 (1990)); the light induciblepromoter from the small subunit of rubisco (Pellegrineschi et al.,Biochem. Soc. Trans. 23(2):247-250 (1995)); the Adh promoter (Walker etal., Proc. Natl. Acad. Sci. U.S.A. 84:6624-66280 (1987)); the sucrosesynthase promoter (Yang et al., Proc. Natl. Acad. Sci. U.S.A.87:4144-4148 (1990)); the R gene complex promoter (Chandler et al.,Plant Cell 1:1175-1183 (1989)); the chlorophyll a/b binding protein genepromoter; and the like.

An angiosperm flower is a complex structure generally consisting of apedicel, sepals, petals, stamens, and a pistil. A stamen comprises afilament and an anther in which the male gametophyte pollens reside. Apistil comprises a stigma, style and ovary. An ovary contains one ormore ovules in which the female gametophyte embryo sac, egg cell,central cell, and other specialized cells reside. Flower promoters ingeneral include promoters that direct gene expression in any of theabove tissues or cell types.

Lipid transfer protein (LTP) genes have been isolated from barley(Federico et al., Plant Mol. Biol. 57:35-51 (2005)), strawberry(Yubero-Serrano et al, J. Exp. Bot. 54:1865-1877 (2003)), Arabidopsis(Thoma et al., Plant Physiol. 105:35-45 (1994)), Norway spruce (Sabalaet al., Plant Mol. Biol. 42:461-478 (2000)), rice (Vignols et al., Gene142:265-270 (1994)), carrot (Toonen et al., Plant J. 12:1213-1221(1997)), Brassica napus (Sohal et al., Plant Mol. Biol. 41:75-87(1999)), Sorghum vulgare (Pelese-Siebenbourg et al., Gene 148:305-308(1994)), and other plant species. The reported LTP genes are known tohave various expression patterns in respective plants. However, thereremains a lack of soybean LTP genes or flower-preferred expression ofLTP genes. LTP assays have been described (Jean-Claude Kader, AnnualReview of Plant Phys. and Plant Mol. Biol. 47: 627-654 (1996). PlantLTPs have eight cysteine residues located at conserved positions. Thecysteine residues are engaged in four disulfide bridges (Jean-ClaudeKader, Annual Review of Plant Phys. and Plant Mol. Biol. 47: 627-654(1996)).

Although advances in technology provide greater success in transformingplants with chimeric genes, there is still a need for preferredexpression of such genes in desired plants. Often times it is desired toselectively express target genes in a specific tissue because oftoxicity or efficacy concerns. For example, flower tissue is a type oftissue where preferred expression is desirable and there remains a needfor promoters that preferably initiate transcription in flower tissue.Promoters that initiate transcription preferably in flower tissuecontrol genes involved in flower development and flower abortion.

SUMMARY OF THE INVENTION

Compositions and methods for regulating gene expression in a plant areprovided. One aspect is for an isolated polynucleotide comprising: a) anucleotide sequence comprising the sequence set forth in SEQ ID NO:1 ora full-length complement thereof; or b) a nucleotide sequence comprisinga sequence having at least 90% sequence identity, based on the BLASTNmethod of alignment, when compared to the sequence set forth in SEQ IDNO:1; wherein said nucleotide sequence is a promoter.

Other embodiments include recombinant DNA constructs comprising apolynucleotide sequence of the present invention operably linked to aheterologous sequence. Additional, some embodiments provide fortransgenic plant cells, transient and stable, transgenic plant seeds, aswell as transgenic plants comprising the provided recombinant DNAconstructs.

There are provided some embodiments that include methods of expressing acoding sequence or a functional RNA in a flowering plant comprising:introducing a recombinant DNA construct described above into the plant,wherein the heterologous sequence comprises a coding sequence; growingthe plant; and selecting a plant displaying expression of the codingsequence or the functional RNA of the recombinant DNA construct.

Furthermore, some embodiments of the present invention include methodsof transgenically altering a marketable flower trait of a floweringplant, comprising: introducing a recombinant DNA construct describedabove into the flowering plant; growing a fertile, mature floweringplant resulting from the introducing step; and selecting a floweringplant expressing the heterologous nucleotide sequence in flower tissuebased on the altered marketable flower trait.

Another aspect is for an isolated polynucleotide comprising: (a) anucleotide sequence encoding a polypeptide, wherein the polypeptide hasat least 80% sequence identity, based on the Clustal method ofalignment, when compared to the sequence set forth in SEQ ID NO:15, or(b) a full-length complement of the nucleotide sequence of (a).

A further aspect is for an isolated polypeptide, wherein the isolatedpolypeptide has at least 80% sequence identity, based on the Clustalmethod of alignment, when compared to the sequence set forth in SEQ IDNO:15.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCES

The invention can be more fully understood from the following detaileddescription, the accompanying drawings and Sequence Listing which form apart of this application. The Sequence Listing contains the one lettercode for nucleotide sequence characters and the three letter codes foramino acids as defined in conformity with the IUPAC-IUBMB standardsdescribed in Nucleic Acids Research 13:3021-3030 (1985) and in theBiochemical Journal 219 (No. 2): 345-373 (1984), which are hereinincorporated by reference in their entirety. The symbols and format usedfor nucleotide and amino acid sequence data comply with the rules setforth in 37 C.F.R. §1.822.

SEQ ID NO:1 is a DNA sequence comprising a 508 bp (base pairs ofnucleotides) soybean LTP4 promoter.

SEQ ID NO:2 is an MPSS tag sequence that is specific to the unique genePSO311306.

SEQ ID NO:3 is a sense primer PSO311306F used in quantitative RT-PCRanalysis of PSO311306 gene expression profile.

SEQ ID NO:4 is an antisense primer PSO311306R used in quantitativeRT-PCR analysis of PSO311306 gene expression profile.

SEQ ID NO:5 is a sense primer ATPS-87F used as an endogenous control ATPsulfurylase gene-specific primer in the quantitative RT-PCR analysis ofPSO311306 gene expression profile.

SEQ ID NO:6 is an antisense primer ATPS-161R used as an endogenouscontrol ATP sulfurylase gene-specific primer in the quantitative RT-PCRanalysis of PSO311306 gene expression profile.

SEQ ID NO:7 is an oligonucleotide primer SAMS-L used in the diagnosticPCR to check for soybean genomic DNA presence in total RNA or cDNA whenpaired with SEQ ID NO:8.

SEQ ID NO:8 is an oligonucleotide primer SAMS-L2 used in the diagnosticPCR to check for soybean genomic DNA presence in total RNA or cDNA whenpaired with SEQ ID NO:7.

SEQ ID NO:9 is the longer strand sequence of the adaptor supplied inClonTech™ GenomeWalker™ kit.

SEQ ID NO:10 is an oligonucleotide primer PSO311306A1 specific to thesoybean PSO311306 gene used in the first nested PCR amplification of theLTP4 promoter when paired with SEQ ID NO:11.

SEQ ID NO:11 is an oligonucleotide primer AP1 used in the first nestedPCR amplification of the LTP4 promoter when paired with SEQ ID NO:10.

SEQ ID NO:12 is an oligonucleotide primer PSO311306A2 specific to thesoybean PSO311306 gene used in the second nested PCR amplification ofthe LTP4 promoter when paired with SEQ ID NO:13. An NcoI restrictionsite CCATGG is added for subsequent cloning.

SEQ ID NO:13 is an oligonucleotide primer AP2 used in the second nestedPCR amplification of the LTP4 promoter when paired with SEQ ID NO:12.

SEQ ID NO:14 is the 669 bp nucleotide sequence of a novel soybean cDNAPSO311306 encoding a polypeptide with similarity to lipid transferproteins. Nucleotides 1 to 55 are the 5′ untranslated sequence,nucleotides 56 to 58 are the translation initiation codon, nucleotides56 to 403 are polypeptide coding region, nucleotides 404 to 406 are thetermination codon, nucleotides 404 to 669 are the 3′ untranslatedsequence.

SEQ ID NO:15 is the 116 amino acid long putative PSO311306 translationproduct LTP4 protein sequence.

SEQ ID NO:16 is a sense primer SAMS-48F used in quantitative PCRanalysis of SAMS:ALS transgene copy numbers.

SEQ ID NO:17 is a FAM labeled fluorescent DNA oligo probe SAMS-88T usedin quantitative PCR analysis of SAMS:ALS transgene copy numbers.

SEQ ID NO:18 is an antisense primer SAMS-134R used in quantitative PCRanalysis of SAMS:ALS transgene copy numbers.

SEQ ID NO:19 is a sense primer YFP-67F used in quantitative PCR analysisof GM-LTP4:YFP transgene copy numbers.

SEQ ID NO:20 is a FAM labeled fluorescent DNA oligo probe YFP-88T usedin quantitative PCR analysis of GM-LTP4:YFP transgene copy numbers.

SEQ ID NO:21 is an antisense primer YFP-130R used in quantitative PCRanalysis of GM-LTP4:YFP transgene copy numbers.

SEQ ID NO:22 is a sense primer used as an endogenous control heat shockprotein gene primer HSP-F1 in quantitative PCR analysis of transgenecopy numbers.

SEQ ID NO:23 is a VIC labeled fluorescent DNA oligo probe used as anendogenous control heat shock protein gene probe HSP in quantitative PCRanalysis of transgene copy numbers.

SEQ ID NO:24 is an antisense primer used as an endogenous control geneheat shock protein primer HSP-R1 in quantitative PCR analysis oftransgene copy numbers.

SEQ ID NO:25 is the 3792 bp sequence of QC372.

SEQ ID NO:26 is the 8317 bp sequence of QC384.

SEQ ID NO:27 is the 8409 bp sequence of QC324i.

SEQ ID NO:28 is the recombination site attL1 sequence in the Gatewaycloning system (Invitrogen™).

SEQ ID NO:29 is the recombination site attL2 sequence in the Gatewaycloning system (Invitrogen™).

SEQ ID NO:30 is the recombination site attR1 sequence in the Gatewaycloning system (Invitrogen™).

SEQ ID NO:31 is the recombination site attR2 sequence in the Gatewaycloning system (Invitrogen™).

SEQ ID NO:32 is the recombination site attB1 sequence in the Gatewaycloning system (Invitrogen™).

SEQ ID NO:33 is the recombination site attB2 sequence in the Gatewaycloning system (Invitrogen™).

FIG. 1 displays the logarithm of relative quantifications of thePSO311306 gene expression in 14 different soybean tissues byquantitative RT-PCR. The gene expression profile indicates that thePSO311306 gene is highly expressed in flower buds and open flowers.

FIG. 2 displays the LTP4 promoter copy number analysis by Southernhybridization.

FIG. 3 is a schematic representation of the map of plasmid QC372,QC3324i, and QC384.

FIG. 4 displays the stable expression of the fluorescent proteinreporter gene ZS-YELLOW1 N1 in the floral and other tissues oftransgenic soybean plants containing a single copy of the transgeneconstruct QC303. The white (green in color display) color indicatesZS-YELLOW1 N1 gene expression. The grey color (red in color display) isbackground auto fluorescence from plant green tissues.

DETAILED DESCRIPTION OF THE INVENTION

The disclosure of all patents, patent applications, and publicationscited herein are incorporated by reference in their entirety.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to “a plant” includes aplurality of such plants, reference to “a cell” includes one or morecells and equivalents thereof known to those skilled in the art, and soforth.

In the context of this disclosure, a number of terms shall be utilized.

The term “promoter” refers to a nucleotide sequence capable ofcontrolling the expression of a coding sequence or functional RNA.Functional RNA includes, but is not limited to, transfer RNA (tRNA) andribosomal RNA (rRNA). Numerous examples of promoters may be found in thecompilation by Okamuro and Goldberg (Biochemistry of Plants 15:1-82(1989)). The promoter sequence consists of proximal and more distalupstream elements, the latter elements often referred to as enhancers.Accordingly, an “enhancer” is a DNA sequence which can stimulatepromoter activity and may be an innate element of the promoter or aheterologous element inserted to enhance the level or tissue-specificityof a promoter. Promoters may be derived in their entirety from a nativegene, or be composed of different elements derived from differentpromoters found in nature, or even comprise synthetic DNA segments. Itis understood by those skilled in the art that different promoters maydirect the expression of a gene in different tissues or cell types, orat different stages of development, or in response to differentenvironmental conditions. Promoters which cause a gene to be expressedin most cell types at most times are commonly referred to as“constitutive promoters”. It is further recognized that, since in mostcases the exact boundaries of regulatory sequences have not beencompletely defined, DNA fragments of some variation may have identicalpromoter activity.

An “intron” is an intervening sequence in a gene that is transcribedinto RNA and then excised in the process of generating the mature mRNA.The term is also used for the excised RNA sequences. An “exon” is aportion of the sequence of a gene that is transcribed and is found inthe mature messenger RNA derived from the gene, and is not necessarily apart of the sequence that encodes the final gene product.

A “flower” is a complex structure consisting of pedicel, sepal, petal,stamen, and carpel. A stamen comprises an anther, pollen and filament. Acarpel comprises a stigma, style and ovary. An ovary comprises an ovule,embryo sac, and egg cell. Soybean pods develop from the pistil. It islikely that a gene expressed in the pistil of a flower continues toexpress in early pod. A “flower cell” is a cell from any one of thesestructures. Flower promoters in general include promoters that directgene expression in any of the above tissues or cell types.

The term “flower crop” or “flowering plants” are plants that produceflowers that are marketable within the floriculture industry. Flowercrops include both cut flowers and potted flowering plants. Cut flowersare plants that generate flowers that can be cut from the plant and canbe used in fresh flower arrangements. Flower crops include roses,carnations, Gerberas, Chrysanthemums, tulips, Gladiolis, Alstroemerias,Anthuriums, lisianthuses, larkspurs, irises, orchids, snapdragons,African violets, azaleas, in addition to other less popular flowercrops.

The terms “flower-specific promoter” or “flower-preferred promoter” maybe used interchangeably herein and refer to promoters active in flower,with promoter activity being significantly higher in flower tissueversus non-flower tissue. “Preferentially initiates transcription”, whendescribing a particular cell type, refers to the relative level oftranscription in that particular cell type as opposed to other celltypes. The described LTP4 promoter is a promoter that preferentiallyinitiates transcription in flower cells. Preferably, the promoteractivity in terms of expression levels of an operably linked sequence ismore than ten-fold higher in flower tissue than non-flower tissue. Morepreferably, the promoter activity is present in flower tissue whileundetectable in non-flower tissue.

As used herein, an “LTP4 promoter” refers to one type of flower-specificpromoter. The native LTP4 promoter (or full-length native LTP4 promoter)is the native promoter of the putative soybean LTP4 polypeptide, whichis a novel soybean protein with homology to many lipid transfer proteinsidentified in other species (see, e.g., Parida and George, Genome50:470-478 (2007); Jaillon et al., Nature 449:463-467 (2007); Finkina etal., Biokhimiia 72:430-438 (2007)). The “LTP4 promoter”, as used herein,also refers to fragments of the full-length native promoter that retainsignificant promoter activity. For example, an LTP4 promoter of thepresent invention can be the full-length promoter (SEQ ID NO:1) or apromoter-functioning fragment thereof. An LTP4 promoter also includesvariants that are substantially similar and functionally equivalent toany portion of the nucleotide sequence set forth in SEQ ID NO:1.

An “isolated nucleic acid fragment” or “isolated polynucleotide” refersto a polymer of ribonucleotides (RNA) or deoxyribonucleotides (DNA) thatis single-stranded or double-stranded, optionally containing synthetic,non-natural or altered nucleotide bases. An isolated polynucleotide inthe form of DNA may be comprised of one or more segments of cDNA,genomic DNA or synthetic DNA.

The terms “polynucleotide”, “polynucleotide sequence”, “nucleic acidsequence”, and “nucleic acid fragment”/“isolated nucleic acid fragment”are used interchangeably herein. These terms encompass nucleotidesequences and the like. A polynucleotide may be a polymer of RNA or DNAthat is single- or double-stranded, that optionally contains synthetic,non-natural or altered nucleotide bases. A polynucleotide in the form ofa polymer of DNA may be comprised of one or more segments of cDNA,genomic DNA, synthetic DNA, or mixtures thereof. Nucleotides (usuallyfound in their 5′-monophosphate form) are referred to by a single letterdesignation as follows: “A” for adenylate or deoxyadenylate (for RNA orDNA, respectively), “C” for cytidylate or deoxycytidylate, “G” forguanylate or deoxyguanylate, “U” for uridylate, “T” fordeoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C orT), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” forany nucleotide.

A “heterologous nucleic acid fragment” or “heterologous nucleotidesequence” refers to a nucleotide sequence that is not naturallyoccurring with the plant promoter sequence of the invention. While thisnucleotide sequence is heterologous to the promoter sequence, it may behomologous, or native, or heterologous, or foreign, to the plant host.However, it is recognized that the instant promoter may be used withtheir native coding sequences to increase or decrease expressionresulting in a change in phenotype in the transformed seed.

The terms “fragment (or variant) that is functionally equivalent” and“functionally equivalent fragment (or variant)” are used interchangeablyherein. These terms refer to a portion or subsequence or variant of thepromoter sequence of the present invention in which the ability toinitiate transcription or drive gene expression (such as to produce acertain phenotype) is retained. Fragments and variants can be obtainedvia methods such as site-directed mutagenesis and syntheticconstruction. As with the provided promoter sequences described herein,the contemplated fragments and variants operate to promote theflower-preferred expression of an operably linked heterologous nucleicacid sequence, forming a recombinant DNA construct (also, a chimericgene). For example, the fragment or variant can be used in the design ofrecombinant DNA constructs to produce the desired phenotype in atransformed plant. Recombinant DNA constructs can be designed for use inco-suppression or antisense by linking a promoter fragment or variantthereof in the appropriate orientation relative to a heterologousnucleotide sequence.

In some aspects of the present invention, the promoter fragments cancomprise at least about 20 contiguous nucleotides, or at least about 50contiguous nucleotides, or at least about 75 contiguous nucleotides, orat least about 100 contiguous nucleotides of SEQ ID NO:1. Thenucleotides of such fragments will usually comprise the TATA recognitionsequence of the particular promoter sequence. Such fragments may beobtained by use of restriction enzymes to cleave the naturally occurringpromoter nucleotide sequences disclosed herein, by synthesizing anucleotide sequence from the naturally occurring promoter DNA sequence,or may be obtained through the use of PCR technology. See particularly,Mullis et al., Methods Enzymol. 155:335-350 (1987), and Higuchi, R. InPCR Technology: Principles and Applications for DNA Amplifications;Erlich, H.A., Ed.; Stockton Press Inc.: New York, 1989.

The terms “substantially similar” and “corresponding substantially” asused herein refer to nucleic acid sequences, particularly promotersequences, wherein changes in one or more nucleotide bases do notsubstantially alter the ability of the promoter to initiatetranscription or drive gene expression or produce a certain phenotype.These terms also refer to modifications, including deletions andvariants, of the nucleic acid sequences of the instant invention by wayof deletion or insertion of one or more nucleotides that do notsubstantially alter the functional properties of the resulting promoterrelative to the initial, unmodified promoter. It is thereforeunderstood, as those skilled in the art will appreciate, that theinvention encompasses more than the specific exemplary sequence.

In one example of substantially similar, substantially similar nucleicacid sequences include those that are also defined by their ability tohybridize to the disclosed nucleic acid sequences, or portions thereof.Substantially similar nucleic acid sequences include those sequencesthat hybridize, under moderately stringent conditions (for example,0.5×SSC, 0.1% SDS, 60° C.) with the sequences exemplified herein, or toany portion of the nucleotide sequences reported herein and which arefunctionally equivalent to the promoter of the invention. Estimates ofsuch homology are provided by either DNA-DNA or DNA-RNA hybridizationunder conditions of stringency as is well understood by those skilled inthe art (Hames and Higgins, Eds.; In Nucleic Acid Hybridisation; IRLPress: Oxford, U.K., 1985). Stringency conditions can be adjusted toscreen for moderately similar fragments, such as homologous sequencesfrom distantly related organisms, to highly similar fragments, such asgenes that duplicate functional enzymes from closely related organisms.Post-hybridization washes partially determine stringency conditions. Oneset of conditions uses a series of washes starting with 6×SSC, 0.5% SDSat room temperature for 15 min, then repeated with 2×SSC, 0.5% SDS at45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50°C. for 30 min. Another set of stringent conditions uses highertemperatures in which the washes are identical to those above except forthe temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS isincreased to 60° C. Another set of highly stringent conditions uses twofinal washes in 0.1×SSC, 0.1% SDS at 65° C.

In some examples, substantially similar nucleic acid sequences are thosesequences that are at least 80% identical to the nucleic acid sequencesreported herein or which are at least 80% identical to any portion ofthe nucleotide sequences reported herein. In some instances,substantially similar nucleic acid sequences are those that are at least90% identical to the nucleic acid sequences reported herein, or at least90% identical to any portion of the nucleotide sequences reportedherein. In some examples, substantially similar nucleic acid sequencesare those that are at least 95% identical to the nucleic acid sequencesreported herein, or are at least 95% identical to any portion of thenucleotide sequences reported herein. It is well understood by oneskilled in the art that many levels of sequence identity are useful inidentifying related polynucleotide sequences. Useful examples of percentidentities are those listed above, or also any integer percentage from80% to 100%, such as, for example, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99%.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without affecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid fragment comprising a nucleotide sequencethat encodes all or a substantial portion of the amino acid sequencesset forth herein. The skilled artisan is well aware of the “codon-bias”exhibited by a specific host cell in usage of nucleotide codons tospecify a given amino acid. Therefore, when synthesizing a nucleic acidsequence for improved expression in a host cell, it is desirable todesign the nucleic acid sequence such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

Sequence alignments and percent similarity calculations may bedetermined using the Megalign program of the LASARGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of thesequences are performed using the Clustal method of alignment (Higginsand Sharp, CABIOS 5:151-153 (1989)) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments and calculation of percent identity of protein sequencesusing the Clustal method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 andDIAGONALS SAVED=5. For nucleic acids these parameters are GAPPENALTY=10, GAP LENGTH PENALTY=10, KTUPLE=2, GAP PENALTY=5, WINDOW=4 andDIAGONALS SAVED=4. A “substantial portion” of an amino acid ornucleotide sequence comprises enough of the amino acid sequence of apolypeptide or the nucleotide sequence of a gene to afford putativeidentification of that polypeptide or gene, either by manual evaluationof the sequence by one skilled in the art, or by computer-automatedsequence comparison and identification using algorithms such as BLAST(Altschul, S. F. et al., J. Mol. Biol. 215:403-410 (1993)) and GappedBlast (Altschul, S. F. et al., Nucleic Acids Res. 25:3389-3402 (1997)).BLASTN refers to a BLAST program that compares a nucleotide querysequence against a nucleotide sequence database.

The term “gene” refers to a nucleic acid fragment that expresses aspecific protein, including regulatory sequences preceding (5′non-coding sequences) and following (3′ non-coding sequences) the codingsequence. “Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” or “recombinant expressionconstruct”, which are used interchangeably, refers to any gene that isnot a native gene, comprising regulatory and coding sequences that arenot found together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, and arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, which is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

“Coding sequence” refers to a DNA sequence that encodes for a specificamino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may include, andare not limited to, promoters, enhancers, translation leader sequences,introns, and polyadenylation recognition sequences.

The “translation leader sequence” refers to a DNA sequence locatedbetween the promoter sequence of a gene and the coding sequence. Thetranslation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner, R. and Foster, G. D., MolecularBiotechnology 3:225 (1995)).

The “3′ non-coding sequences” refer to DNA sequences located downstreamof a coding sequence and include polyadenylation recognition sequencesand other sequences encoding regulatory signals capable of affectingmRNA processing or gene expression. The polyadenylation signal isusually characterized as affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al., Plant Cell1:671-680 (1989).

“RNA transcript” refers to a product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When an RNAtranscript is a perfect complementary copy of a DNA sequence, it isreferred to as a primary transcript, or it may be a RNA sequence derivedfrom posttranscriptional processing of a primary transcript and isreferred to as a mature RNA. “Messenger RNA” (“mRNA”) refers to RNA thatis without introns and that can be translated into protein by the cell.“cDNA” refers to a DNA that is complementary to and synthesized from anmRNA template using the enzyme reverse transcriptase. The cDNA can besingle-stranded or converted into the double-stranded using the Klenowfragment of DNA polymerase I. “Sense” RNA refers to RNA transcript thatincludes mRNA and so can be translated into protein within a cell or invitro. “Antisense RNA” refers to a RNA transcript that is complementaryto all or part of a target primary transcript or mRNA and that blocksexpression or transcripts accumulation of a target gene. Thecomplementarity of an antisense RNA may be with any part of the specificgene transcript, i.e. at the 5′ non-coding sequence, 3′ non-codingsequence, introns, or the coding sequence. “Functional RNA” refers toantisense RNA, ribozyme RNA, or other RNA that may not be translated yethas an effect on cellular processes.

The term “operably linked” refers to the association of nucleic acidsequences on a single polynucleotide so that the function of one isaffected by the other. For example, a promoter is operably linked with aheterologous nucleotide sequence, e.g., a coding sequence, when it iscapable of affecting the expression of that heterologous nucleotidesequence (i.e., for example, the coding sequence is under thetranscriptional control of the promoter). A coding sequence can beoperably linked to promoter sequences in sense or antisense orientation.

The terms “initiate transcription”, “initiate expression”, “drivetranscription”, and “drive expression” are used interchangeably hereinand all refer to the primary function of a promoter. As detailedthroughout this disclosure, a promoter is a non-coding genomic DNAsequence, usually upstream (5′) to the relevant coding sequence, and itsprimary function is to act as a binding site for RNA polymerase andinitiate transcription by the RNA polymerase. Additionally, there is“expression” of RNA, including functional RNA, or the expression ofpolypeptide for operably linked encoding nucleotide sequences, as thetranscribed RNA ultimately is translated into the correspondingpolypeptide.

The term “expression”, as used herein, refers to the production of afunctional end-product, e.g., an mRNA or a protein (precursor ormature).

The term “recombinant DNA construct” or “recombinant expressionconstruct” is used interchangeably and refers to a discretepolynucleotide into which a nucleic acid sequence or fragment can bemoved. Preferably, it is a plasmid vector or a fragment thereofcomprising the promoters of the present invention. The choice of plasmidvector is dependent upon the method that will be used to transform hostplants. The skilled artisan is well aware of the genetic elements thatmust be present on the plasmid vector in order to successfullytransform, select and propagate host cells containing the recombinantDNA construct. The skilled artisan will also recognize that differentindependent transformation events will result in different levels andpatterns of expression (Jones et al., EMBO J. 4:2411-2418 (1985); DeAlmeida et al., Mol. Gen. Genetics 218:78-86 (1989)), and thus thatmultiple events must be screened in order to obtain lines displaying thedesired expression level and pattern. Such screening may be accomplishedby PCR and Southern analysis of DNA, RT-PCR and Northern analysis ofmRNA expression, Western analysis of protein expression, or phenotypicanalysis.

Expression or overexpression of a gene involves transcription of thegene and translation of the mRNA into a precursor or mature protein.“Antisense inhibition” refers to the production of antisense RNAtranscripts capable of suppressing the expression of the target protein.“Overexpression” refers to the production of a gene product intransgenic organisms that exceeds levels of production in normal ornon-transformed organisms. “Co-suppression” refers to the production ofsense RNA transcripts capable of suppressing the expression ortranscript accumulation of identical or substantially similar foreign orendogenous genes (U.S. Pat. No. 5,231,020). The mechanism ofco-suppression may be at the DNA level (such as DNA methylation), at thetranscriptional level, or at post-transcriptional level.

Co-suppression constructs in plants previously have been designed byfocusing on overexpression of a nucleic acid sequence having homology toan endogenous mRNA, in the sense orientation, which results in thereduction of all RNA having homology to the overexpressed sequence(Vaucheret et al., Plant J. 16:651-659 (1998); and Gura, Nature404:804-808 (2000)). The overall efficiency of this phenomenon is low,and the extent of the RNA reduction is widely variable. Recent work hasdescribed the use of “hairpin” structures that incorporate all, or part,of an mRNA encoding sequence in a complementary orientation that resultsin a potential “stem-loop” structure for the expressed RNA (PCTPublication Nos. WO99/53050 and WO02/00904). This increases thefrequency of co-suppression in the recovered transgenic plants. Anothervariation describes the use of plant viral sequences to direct thesuppression, or “silencing”, of proximal mRNA encoding sequences (PCTPublication No. WO98/36083). Neither of these co-suppressing phenomenahas been elucidated mechanistically at the molecular level, althoughgenetic evidence has been obtained that may lead to the identificationof potential components (Elmayan et al., Plant Cell 10:1747-1757(1998)).

As stated herein, “suppression” refers to a reduction of the level ofenzyme activity or protein functionality (e.g., a phenotype associatedwith a protein) detectable in a transgenic plant when compared to thelevel of enzyme activity or protein functionality detectable in anon-transgenic or wild type plant with the native enzyme or protein. Thelevel of enzyme activity in a plant with the native enzyme is referredto herein as “wild type” activity. The level of protein functionality ina plant with the native protein is referred to herein as “wild type”functionality. The term “suppression” includes lower, reduce, decline,decrease, inhibit, eliminate and prevent. This reduction may be due to adecrease in translation of the native mRNA into an active enzyme orfunctional protein. It may also be due to the transcription of thenative DNA into decreased amounts of mRNA and/or to rapid degradation ofthe native mRNA. The term “native enzyme” refers to an enzyme that isproduced naturally in a non-transgenic or wild type cell. The terms“non-transgenic” and “wild type” are used interchangeably herein.

“Altering expression” refers to the production of gene product(s) intransgenic organisms in amounts or proportions that differ significantlyfrom the amount of the gene product(s) produced by the correspondingwild-type organisms (i.e., expression is increased or decreased).

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. Thus, a “transgenicplant cell” as used herein refers to a plant cell containing thetransformed nucleic acid fragments. The preferred method of soybean celltransformation is use of particle-accelerated or “gene gun”transformation technology (Klein et al., Nature (London) 327:70-73(1987); U.S. Pat. No. 4,945,050).

“Transient expression” refers to the temporary expression of oftenreporter genes such as β-glucuronidase (GUS), fluorescent protein genesGFP, ZS-YELLOW1 N1, AM-CYAN1, DS-RED in selected certain cell types ofthe host organism in which the transgenic gene is introduced temporallyby a transformation method. The transformed materials of the hostorganism are subsequently discarded after the transient gene expressionassay.

A “marketable flower trait” is a characteristic or phenotype of theflower of a plant such as the color, scent or morphology of a flower.The marketable flower trait is a characteristic of a flower that is ofhigh regard to a flower crop consumer in deciding whether to purchasethe flower crop.

The phrase “genes involved in anthocyanin biosynthesis” refers to genesthat encode proteins that play a role in converting metabolic precursorsinto the one of a number of anthocyanins. Examples of genes involved inthe biosynthesis of anthocyanin are dyhydroflavonol 4-reductase,flavonoid 3,5-hydroxylase, chalcone synthase, chalcone isomerase,flavonoid 3-hydroxylase, anthocyanin synthase, and UDP-glucose3-O-flavonoid glucosyl transferase (see, e.g., Mori et al., Plant CellReports 22:415-421 (2004)).

The phrase “genes involved in the biosynthesis of fragrant fatty acidderivatives” refers to genes that encode proteins that play a role inmanipulating the biosynthesis of fragrant fatty acid derivatives such asterpenoids, phenylpropanoids, and benzenoids in flowers (see, e.g.,Tanaka et al., Plant Cell, Tissue and Organ Culture 80:1-24 (2005)).Examples of such genes include S-linalool synthase, acetylCoA:benzylalcohol acetyltransferase, benzyl CoA:benzylalcohol benzoyltransferase, S-adenosyl-L-methionine:benzoic acid carboxyl methyltransferase (BAMT), mycrene synthases, (E)-β-ocimene synthase, orcinolO-methyltransferase, and limonene synthases (see, e.g., Tanaka et al.,supra).

The term “flower homeotic genes” or “flower morphology modifying genes”refers to genes that are involved in pathways associated with flowermorphology. A modification of flower morphology can lead to a novel formof the respective flower that can enhance its value in the flower cropmarketplace. Morphology can include the size, shape, or petal pattern ofa flower. Some example of flower homeotic genes include genes involvedin cell-fate determination (in ABC combinatorial model of geneexpression), including AGAMOUS, which determines carpel fate in thecentral whorl, APETALA3, which determines the sepal fate in the outerwhorl, and PISTILLATA, which determines petal development in the secondwhorl (Espinosa-Soto et al., Plant Cell 16:2923-2939 (2004)).

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook, J.et al., In Molecular Cloning: A Laboratory Manual; 2^(nd) ed.; ColdSpring Harbor Laboratory Press: Cold Spring Harbor, N.Y., 1989(hereinafter “Sambrook et al., 1989”) or Ausubel, F. M., Brent, R.,Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. and Struhl,K., Eds.; In Current Protocols in Molecular Biology; John Wiley andSons: New York, 1990 (hereinafter “Ausubel et al., 1990”).

“PCR” or “Polymerase Chain Reaction” is a technique for the synthesis oflarge quantities of specific DNA segments consisting of a series ofrepetitive cycles (Perkin Elmer Cetus Instruments, Norwalk, Conn.).Typically, the double stranded DNA is heat denatured; the two primerscomplementary to the 3′ boundaries of the target segment are annealed atlow temperature and then extended at an intermediate temperature. Oneset of these three consecutive steps comprises a cycle.

Embodiments of the present invention include isolated polynucleotidescomprising a nucleotide sequence that is a promoter. In some instancesthe nucleotide sequence includes one or more of the following:

-   -   a) the sequence set forth in SEQ ID NO:1 or a full-length        complement thereof; or    -   b) a nucleotide sequence comprising a sequence having at least        90% sequence identity, based on the BLASTN method of alignment,        when compared to the sequence set forth in SEQ ID NO:1.

In other aspects, the nucleotide sequence includes one or more of thefollowing:

-   -   (a) a nucleotide sequence comprising a fragment of SEQ ID NO:1,        or    -   (b) a nucleotide sequence comprising a sequence having at least        90% sequence identity, based on the BLASTN method of alignment,        when compared to the nucleotide sequence of (a).        The nucleotide sequences of the present invention can be        referred to as a promoter or as having promoter-like activity.        In some embodiments the nucleotide sequence is a promoter that        preferentially initiates transcription in a plant flower cell.        Such promoter is referred to as a flower-specific promoter.        Preferably the promoter of the present invention is the soybean        “LTP4” promoter. The LTP4 promoter of the invention expresses in        stigma, therefore the promoter may be used to express genes        involved in pollination compatibility.

In a preferred embodiment, the promoter comprises the nucleotidesequence set forth in SEQ ID NO:1. The present invention also includesnucleic acid fragments, variants, and complements of the aforementionednucleotide sequences or promoters, provided that they are substantiallysimilar and functionally equivalent to the nucleotide sequence set forthin these nucleotide sequences. A nucleic acid fragment or variant thatis functionally equivalent to the present LTP4 promoter is any nucleicacid fragment or variant that is capable of initiating the expression,preferably initiating flower-specific expression, of a coding sequenceor functional RNA in a similar manner to the LTP4 promoter. Theexpression patterns of LTP4 gene and its promoter are set forth inExamples 1, 2, and 6.

In some aspects, a recombinant DNA construct can be formed in part byoperably linking at least one of the promoters of the present inventionto any heterologous nucleotide sequence. The heterologous nucleotidesequence can be expressed in a cell as either a functional RNA or apolypeptide. The cell for expression includes a plant or bacterial cell,preferably a plant cell. The recombinant DNA construct preferablyincludes the LTP4 promoter. The recombinant DNA construct preferablyincludes a heterologous nucleotide sequence that encodes a protein thatplays a role in flower color formation, fragrance production, orshape/morphology development of the flower. The color of a flower can bealtered transgenically by expressing genes involved in betalain,carotenoid, or flavanoid biosynthesis. In regard to genes involved inthe biosynthesis of anthocyanin, dyhydroflavonol 4-reductase, flavonoid3,5-hydroxylase, chalcone synthase, chalcone isomerase, flavonoid3-hydroxylase, anthocyanin synthase, and UDP-glucose 3-O-flavonoidglucosyl transferase are some examples. The scent of a flower can bealtered transgenically by expressing genes that manipulate thebiosynthesis of fragrant fatty acid derivatives such as terpenoids,phenylpropanoids, and benzenoids in flowers. Some embodiments of theinvention include a heterologous nucleotide sequence that is selectedfrom S-linalool synthase, acetyl CoA:benzylalcohol acetyltransferase,benzyl CoA:benzylalcohol benzoyl transferase,S-adenosyl-L-methionine:benzoic acid carboxyl methyl transferase,mycrene synthases, (E)-β-ocimene synthase, orcinol O-methyltransferase,or limonene synthases. Flower structures/morphologies can be alteredtransgenically by expressing flower homeotic genes to create novelornamental varieties. Some embodiments of the invention include aheterologous nucleotide sequence that is selected from genes such as,for example, AGAMOUS, APETALA3, and PISTILLATA.

It is recognized that the instant promoters may be used with theirnative coding sequences to increase or decrease expression in flowertissue. The selection of the heterologous nucleic acid fragment dependsupon the desired application or phenotype to be achieved. The variousnucleic acid sequences can be manipulated so as to provide for thenucleic acid sequences in the proper orientation.

Plasmid vectors comprising the instant recombinant DNA construct can beconstructed. The choice of plasmid vector is dependent upon the methodthat will be used to transform host cells. The skilled artisan is wellaware of the genetic elements that must be present on the plasmid vectorin order to successfully transform, select and propagate host cellscontaining the recombinant DNA construct.

The described polynucleotide embodiments encompass isolated orsubstantially purified nucleic acid compositions. An “isolated” or“purified” nucleic acid molecule, or biologically active portionthereof, is substantially free of other cellular material or culturemedium when produced by recombinant techniques, or substantially free ofchemical precursors or other chemicals when chemically synthesized. An“isolated” nucleic acid is essentially free of sequences (preferablyprotein encoding sequences) that naturally flank the polynucleotide(i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) inthe genomic DNA of the organism from which the polynucleotide isderived. For example, in various embodiments, the isolatedpolynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb,0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank thepolynucleotide in genomic DNA of the cell from which the polynucleotideis derived.

In another embodiment, the present invention includes host cellscomprising either the recombinant DNA constructs or isolatedpolynucleotides of the present invention. Examples of the host cells ofthe present invention include, and are not limited to, yeast, bacteria,and plants, including flower crops such as, e.g., rose, carnation,Gerbera, Chrysanthemum, tulip, Gladioli, Alstroemeria, Anthurium,lisianthus, larkspur, irises, orchid, snapdragon, African violet, orazalea. Preferably, the host cells are plant cells, and more preferably,flower crop cells, and more preferably, Gerbera, rose, carnation,Chrysanthemum, or tulip cells.

Methods for transforming dicots, primarily by use of Agrobacteriumtumefaciens, and obtaining transgenic plants have been published, amongothers, for cotton (U.S. Pat. No. 5,004,863, U.S. Pat. No. 5,159,135);soybean (U.S. Pat. No. 5,569,834, U.S. Pat. No. 5,416,011); Brassica(U.S. Pat. No. 5,463,174); peanut (Cheng et al., Plant Cell Rep.15:653-657 (1996); McKently et al., Plant Cell Rep. 14:699-703 (1995));papaya (Ling et al., Bio/technology 9:752-758 (1991)); and pea (Grant etal., Plant Cell Rep. 15:254-258 (1995)). For a review of other commonlyused methods of plant transformation see Newell, C. A., Mol. Biotechnol.16:53-65 (2000). One of these methods of transformation usesAgrobacterium rhizogenes (Tepfler, M. and Casse-Delbart, F., Microbiol.Sci. 4:24-28 (1987)). Transformation of soybeans using direct deliveryof DNA has been published using PEG fusion (PCT Publication No. WO92/17598), electroporation (Chowrira et al., Mol. Biotechnol. 3:17-23(1995); Christou et al., Proc. Natl. Acad. Sci. U.S.A. 84:3962-3966(1987)), microinjection (Neuhaus et al., Physiol. Plant. 79:213-217(1990)), or particle bombardment (McCabe et al., Biotechnology 6:923(1988); Christou et al., Plant Physiol. 87:671-674 (1988)).

In another embodiment, the present invention includes transgenic plantscomprising the recombinant DNA constructs provided herein. Thetransgenic plants are selected from, for example, one of a number ofvarious flower crops including roses, carnations, Gerberas,Chrysanthemums, tulips, Gladiolis, Alstroemerias, Anthuriums,lisianthuses, larkspurs, irises, orchids, snapdragons, African violets,azaleas, in addition to other less popular flower crops.

In some embodiments of the invention, there are provided transgenicseeds produced by the transgenic plants provided. Such seeds are able toproduce another generation of transgenic plants.

There are a variety of methods for the regeneration of plants from planttissues. The particular method of regeneration will depend on thestarting plant tissue and the particular plant species to beregenerated. The regeneration, development and cultivation of plantsfrom single plant protoplast transformants or from various transformedexplants is well known in the art (Weissbach and Weissbach, Eds.; InMethods for Plant Molecular Biology; Academic Press, Inc.: San Diego,Calif., 1988). This regeneration and growth process typically includesthe steps of selection of transformed cells, culturing thoseindividualized cells through the usual stages of embryonic developmentthrough the rooted plantlet stage. Transgenic embryos and seeds aresimilarly regenerated. The resulting transgenic rooted shoots arethereafter planted in an appropriate plant growth medium such as soil.Preferably, the regenerated plants are self-pollinated to providehomozygous transgenic plants. Otherwise, pollen obtained from theregenerated plants is crossed to seed-grown plants of agronomicallyimportant lines. Conversely, pollen from plants of these important linesis used to pollinate regenerated plants. A transgenic plant of thepresent invention containing a desired polypeptide is cultivated usingmethods well known to one skilled in the art.

In addition to the above discussed procedures, there are generallyavailable standard resource materials that describe specific conditionsand procedures for the construction, manipulation and isolation ofmacromolecules (e.g., DNA molecules, plasmids, and the like), generationof recombinant DNA fragments and recombinant expression constructs, andthe screening and isolating of clones (see, for example, Sambrook etal., 1989; Maliga et al., In Methods in Plant Molecular Biology; ColdSpring Harbor Press, 1995; Birren et al., In Genome Analysis: DetectingGenes, 1; Cold Spring Harbor: New York, 1998; Birren et al., In GenomeAnalysis: Analyzing DNA, 2; Cold Spring Harbor: New York, 1998; Clark,Ed., In Plant Molecular Biology: A Laboratory Manual; Springer: NewYork, 1997).

The skilled artisan will also recognize that different independenttransformation events will result in different levels and patterns ofexpression of the chimeric genes (Jones et al., EMBO J. 4:2411-2418(1985); De Almeida et al., Mol. Gen. Genetics 218:78-86 (1989)). Thus,multiple events must be screened in order to obtain lines displaying thedesired expression level and pattern. Such screening may be accomplishedby northern analysis of mRNA expression, western analysis of proteinexpression, or phenotypic analysis. Also of interest are seeds obtainedfrom transformed plants displaying the desired expression profile.

The level of activity of the LTP4 promoter in flowers is in some casescomparable to that of many known strong promoters such as the CaMV 35Spromoter (Atanassova et al., Plant Mol. Biol. 37:275-285 (1998); Battrawand Hall, Plant Mol. Biol. 15:527-538 (1990); Holtorf et al., Plant Mol.Biol. 29:637-646 (1995); Jefferson et al., EMBO J. 6:3901-3907 (1987);Wilmink et al., Plant Mol. Biol. 28:949-955 (1995)), the Arabidopsisoleosin promoters (Plant et al., Plant Mol. Biol. 25:193-205 (1994); Li,Texas A&M University Ph.D. dissertation, pp. 107-128 (1997)), theArabidopsis ubiquitin extension protein promoters (Callis et al., J.Biol. Chem. 265(21):12486-12493 (1990)), a tomato ubiquitin genepromoter (Rollfinke et al., Gene 211:267-276 (1998)), a soybean heatshock protein promoter (Raschke et al., J. Mol. Biol. 199(4):549-557(1988)), and a maize H3 histone gene promoter (Atanassova et al., PlantMol. Biol. 37:275-285 (1998)).

In some embodiments, the promoters of the present invention are usefulwhen flower-specific expression of a target heterologous nucleic acidfragment is required. Another useful feature of the promoters is itsexpression profile having high levels in developing stigmas (See Example6). The promoters of the present invention are most active in thestigmas of developing flower buds and open flowers. Thus, the promoterscan be used for gene expression or gene silencing in flowers, especiallywhen gene expression or gene silencing is desired predominantly instigmas.

In some embodiments, the promoters of the present invention are toconstruct recombinant DNA constructs that can be used to reduceexpression of at least one heterologous nucleic acid sequence in a plantcell. To accomplish this, a recombinant DNA construct can be constructedby linking the heterologous nucleic acid sequence to a promoter of thepresent invention. (See U.S. Pat. No. 5,231,020 and PCT PublicationsWO99/53050, WO02/00904, and WO98/36083 for methodology to block plantgene expression via cosuppression) Alternatively, recombinant DNAconstructs designed to express antisense RNA for a heterologous nucleicacid fragment can be constructed by linking the fragment in reverseorientation to a promoter of the present invention. (See U.S. Pat. No.5,107,065 for methodology to block plant gene expression via antisenseRNA) Either the cosuppression or antisense chimeric gene can beintroduced into plants via transformation. Transformants, whereinexpression of the heterologous nucleic acid sequence is decreased oreliminated, are then selected.

There are embodiments of the present invention that include promoters ofthe present invention being utilized for methods of altering (increasingor decreasing) the expression of at least one heterologous nucleic acidsequence in a plant cell which comprises: transforming a plant cell witha recombinant DNA expression construct described herein; growing fertilemature plants from the transformed plant cell; and selecting plantscontaining a transformed plant cell wherein the expression of theheterologous nucleotide sequence is altered (increased or decreased).

Transformation and selection can be accomplished using methodswell-known to those skilled in the art including, but not limited to,the methods described herein.

There are provided some embodiments that include methods of expressing acoding sequence in a plant that is a flower crop comprising: introducinga recombinant DNA construct disclosed herein into the plant; growing theplant; and selecting a plant displaying expression of the codingsequence; wherein the nucleotide sequence comprises: a nucleotidesequence comprising the sequence set forth in SEQ ID NO:1 or afull-length complement thereof; a nucleotide sequence comprising afragment of the sequence set forth in SEQ ID NO:1, or a nucleotidesequence comprising a sequence having at least 90% sequence identity,based on the BLASTN method of alignment, when compared to the sequenceset forth in SEQ ID NO:1; wherein said nucleotide sequence initiatestranscription in a flower cell of the plant.

Furthermore, some embodiments of the present invention include methodsof transgenically altering a marketable flower trait of a floweringplant, comprising: introducing a recombinant DNA construct disclosedherein into the flowering plant; growing a fertile, mature floweringplant resulting from the introducing step; and selecting a floweringplant expressing the heterologous nucleotide sequence in flower tissuebased on the altered marketable flower trait.

As further described in the Examples below, the promoter activity of thesoybean genomic DNA fragment sequence SEQ ID NO:1 upstream of the LTP4protein coding sequence was assessed by linking the fragment to a yellowfluorescence reporter gene, ZS-YELLOW1 N1 (YFP) (Matz et al., Nat.Biotechnol. 17:969-973 (1999)), transforming the promoter::YFPexpression cassette into soybean, and analyzing YFP expression invarious cell types of the transgenic plants (see Example 6). All partsof the transgenic plants were analyzed and YFP expression waspredominantly detected in flowers, and more specifically in stigmas.These results indicated that the nucleic acid fragment containedflower-preferred promoter.

Some embodiments of the present invention provide recombinant DNAconstructs comprising at least one isopentenyl transferase nucleic acidsequence operably linked to a provide promoter, preferably a LTP4promoter. The isopentenyl transferase plays a key step in thebiosynthesis of plant cytokinin (Kakimoto, J. Plant Res. 116:233-239(2003)). Elevated levels of cytokinin in plant cells might help to delayfloral senescence and abortion which may present a potential way toimprove crop yields (Chang et al., Plant Physiol. 132:2174-2183 (2003);Young et al., Plant J. 38:910-922 (2004)).

Utilities for Flower-Specific Promoters

The color, scent or morphology of a flower represents marketable flowertraits, or characteristics/phenotypes of a flower that consumers,particularly floriculturalists, consider when determining which flowersare desirable and will be purchased. Hence, it would be beneficial to beable to alter these characteristics in order to satisfy the desires ofconsumers. Transgenic technologies can be implemented in order toachieve such results.

The phenotype of a flower can be altered transgenically by expressinggenes, preferably in flower tissue, that play a role in color formation,fragrance production, or shape/morphology development of the flower.This type of alteration is particularly useful in the floricultureindustry, and particularly useful for flowering plants.

The color of a flower is mainly the result of three types of pigment,flavanoids, carotenoids, and betalains. The flavanoids are the mostcommon of the three and they contribute to colors ranging from yellow tored to blue, with anthocyanins being the major flavanoid. Carotenoidsare C-40 tetraterpenoids that contribute to the majority of yellow huesand contribute to orange/red, bronze and brown colors, e.g., that seenin roses and chrysanthemums. Betalains are the least abundant andcontribute to various hues of ivory, yellow, orange, red and violet. Thecolor of a flower can be altered transgenically by expressing genesinvolved in, e.g., betalain, carotenoid, or flavanoid biosynthesis. Inone example, the color of a flower can be altered transgenically byexpressing genes involved in the biosynthesis of anthocyanin, forexample, dyhydroflavonol 4-reductase, flavonoid 3,5-hydroxylase,chalcone synthase, chalcone isomerase, flavonoid 3-hydroxylase,anthocyanin synthase, and UDP-glucose 3-O-flavonoid glucosyltransferase. In some aspects of the invention, the gene involved inanthocyanin biosynthesis is the flavonoid 3,5-hydroxylase gene (see,e.g., Mori et al., Plant Cell Reports 22:415-421 (2004)). This type ofalteration is particularly useful in the floriculture industry,providing novel flower colors in flower crops.

In addition to color, the scent of a flower can be alteredtransgenically by expressing genes that manipulate the biosynthesis offragrant fatty acid derivatives such as terpenoids, phenylpropanoids,and benzenoids in flowers (see, e.g., Tanaka et al., Plant Cell, Tissueand Organ Culture 80:1-24 (2005)). Genes involved in the biosynthesis offragrant fatty acid derivatives can be operably linked to theflower-specific promoters presently described for preferentialexpression in flower tissue. The preferential expression in flowertissue can be utilized to generate new and desirable fragrances toenhance the demand for the underlying cut flower. A number of knowngenes that are involved in the biosynthesis of floral scents aredescribed below. A strong sweet scent can be generated in a flower byintroducing or up-regulating expression of S-linalool synthase, whichwas earlier isolated from Clarkia breweri. Two genes that areresponsible for the production of benzylacetate and benzylbenzoate areacetyl CoA:benzylalcohol acetyltransferase and benzyl CoA:benzylalcoholbenzoyl transferase, respectively. These transferases were also reportedto have been isolated from C. breweri. A phenylpropanoid floral scent,methylbenzoate, is synthesized in part byS-adenosyl-L-methionine:benzoic acid carboxyl methyl transferase (BAMT),which catalyzes the final step in the biosynthesis of methyl benzoate.BAMT is known to have a significant role in the emission of methylbenzoate in snapdragon flowers. Two monoterpenes, mycrene and(E)-β-ocimene, from snapdragon are known to be synthesized in part bythe terpene synthases: mycrene synthases and (E)-β-ocimene synthases.Other genes involved in biosynthesis of floral scents have been reportedand are being newly discovered, many of which are isolated from rose.Some genes involved in scent production in the rose include orcinolO-methyltransferase, for synthesis of S-adenosylmethionine, and limonenesynthases (see, e.g., Tanaka et al., supra).

Flower structures/morphologies can be altered transgenically byexpressing flower homeotic genes to create novel ornamental varieties.The flower homeotic genes that are determinative of flower morphologyinclude genes such as AGAMOUS, APETALA3, PISTILLATA, and others that areknown and/or are being elucidated (see, e.g., Espinosa-Soto et al.,Plant Cell 16:2923-2939 (2004)).

EXAMPLES

Aspects of the present invention are exemplified in the followingExamples. It should be understood that these Examples, while indicatingpreferred embodiments of the invention, are given by way of illustrationonly. From the above discussion and these Examples, one skilled in theart can ascertain the essential characteristics of this invention, andwithout departing from the spirit and scope thereof, can make variouschanges and modifications of the invention to adapt it to various usagesand conditions. Thus, various modifications of the invention in additionto those shown and described herein will be apparent to those skilled inthe art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

In the discussion below, parts and percentages are by weight and degreesare Celsius, unless otherwise stated. Sequences of promoters, cDNA,adaptors, and primers listed herein are in the 5′ to 3′ orientationunless described otherwise. Techniques in molecular biology weretypically performed as described in Ausubel et al., 1990 or Sambrook etal., 1989.

Example 1 Lynx MPSS Profiling of Soybean Genes Preferably Expressed inFlowers

Soybean expression sequence tags (ESTs) were generated by sequencingrandomly selected clones from cDNA libraries constructed from differentsoybean tissues. Multiple EST sequences may have different lengthsrepresenting different regions of the same soybean gene. For those ESTsequences representing the same gene that are found more frequently in aflower-specific cDNA library, there is a possibility that therepresentative gene could be a flower preferred gene candidate. MultipleEST sequences representing the same soybean gene were compiledelectronically based on their overlapping sequence homology into a fulllength sequence representing a unique gene. These assembled, unique genesequences were cumulatively collected and the information was stored ina searchable database. Flower specific candidate genes were identifiedby searching this database to find gene sequences that are frequentlyfound in flower libraries but are rarely found in other tissuelibraries, or not found in other tissue libraries.

One unique gene, PSO311306, was identified in the search as a flowerspecific gene candidate since all of the ESTs representing PSO311306were mostly found in flower tissue. PSO311306 cDNA sequence (SEQ IDNO:14) as well as its putative translated protein sequence (SEQ IDNO:15) were used to search National Center for Biotechnology Information(NCBI) databases. PSO311306 was found to represent a novel soybean genewith significant homology to lipid transfer protein genes identified indifferent species (e.g., over 50% identity to lipid transfer proteinsfrom, e.g., Retama raetam (white weepin broom), Prosopis juliflora(Mesquite), Vitis vinifera (European grapevine), Davidia involucratav(dove tree), Prunus avium (sweet cherry), Populus alba x Populus tremulavar. glandulosa (poplar), Prunus persica (peach), Vigna radiate (mungbean)). PSO311306 was subsequently named LTP4 to reflect this sequencehomology.

A more sensitive gene expression profiling methodology MPSS (MassParallel Signature Sequence) transcript profiling technique (Brenner etal., Proc Natl Acad Sci USA 97:1665-70 (2000)) was used to confirmPSO311306 as a flower specific gene. The MPSS technology involves thegeneration of 17 base signature tags from mRNA samples that have beenreverse transcribed from poly A+ RNA isolated using standard molecularbiology techniques (Sambrook et al., 1989). The tags are simultaneouslysequenced and assigned to genes or ESTs. The abundance of these tags isgiven a number value that is normalized to parts per million (PPM) whichthen allows the tag expression, or tag abundance, to be compared acrossdifferent tissues. Genome wide gene expressions can be profiledsimultaneously using this technology. Since each 17 base tag is longenough to be specific to only one or a few genes in any genome, the MPSSplatform can be used to determine the expression pattern of a particulargene and its expression levels in different tissues.

MPSS gene expression profiles were generated from different soybeantissues over time, and the profiles were accumulated in a searchabledatabase. PSO311306 cDNA sequence SEQ ID NO:14 was used to search theMPSS database to identify a MPSS tag sequence (SEQ ID NO:2) that isidentical to a 17 base pair region from position 375 to 391 at the endof PSO311306 polypeptide coding region. The identified MPSS tag was thenused to search the MPSS database to reveal its abundance in differentsoybean tissues. As illustrated in Table 1, the PSO311306 gene wasconfirmed to be highly abundant in flowers and also detectable in leaf,pod, and petiole, a desired expression profile for its promoter to beable to express genes in flowers.

TABLE 1 Lynx MPSS Expression Profiles of the PSO311306 Gene Target genePSO311306 Tag sequence SEQ ID NO: 2 Flower 7556 Pod 201 Flower bud 10064Lateral root 0 Leaf 445 Petiole 160 Primary root 0 Seed 0 Stem 12

Example 2 Quantitative RT-PCR Profiles of LTP4 Gene Expression inSoybean

The MPSS profiles of LTP4 gene, i.e. PSO311306, was confirmed andextended by analyzing 14 different soybean tissues using the relativequantitative RT-PCR (qRT-PCR) technique with a 7500 real time PCR system(Applied Biosystems, Foster City, Calif.).

Fourteen soybean tissues (somatic embryo in suspension culture, somaticembryo grown one week on solid medium, leaf, leaf petiole, root, flowerbud, open flower, R3 pod, R4 seed, R4 pod coat, R5 seed, R5 pod coat, R6seed, R6 pod coat) were collected from cultivar ‘Jack’ and flash frozenin liquid nitrogen. The seed and pod development stages were definedaccording to descriptions in Fehr and Caviness, IWSRBC 80:1-12 (1977).Total RNA was extracted with Trizol reagents (Invitrogen™, Carlsbad,Calif.) and treated with DNase I to remove any trace amount of genomicDNA contamination. The first strand cDNA was synthesized withSuperscript III reverse transcriptase (Invitrogen™).

PCR analysis was performed to confirm that the cDNA was free of genomicDNA. The forward and reverse primers used for the PCR analysis are shownin SEQ ID NO:7 and SEQ ID NO:8. The primers are specific to the 5′UTRintron/exon junction region of a soybean S-adenosylmethionine synthetase(SAMS) gene promoter (PCT Publication No. WO00/37662). PCR using thisprimer set will amplify a 967 bp DNA fragment from a soybean genomic DNAtemplate and a 376 bp DNA fragment from the cDNA template. The genomicDNA-free cDNA aliquots were used in qRT-PCR analysis of PSO311306 usinggene-specific primers SEQ ID NO:3 and SEQ ID NO:4. An endogenous soybeanATP sulfurylase gene was used as an internal control for normalizationwith primers SEQ ID NO:5 and SEQ ID NO:6 and soybean wild type genomicDNA was used as the calibrator for relative quantification.

The qRT-PCR profiling of the LTP4 gene expression confirmed itspredominant flower expression and also showed ongoing expression inyoung R3 pod and R4 pod coat (FIG. 1).

Example 3 Isolation of Soybean LTP4 Promoter

The soybean genomic DNA fragment corresponding to the LTP4 promoter wasisolated using a polymerase chain reaction (PCR) based approach calledgenome walking using the Universal GenomeWalker™ kit from Clontech™(Product User Manual No. PT3042-1).

Soybean genomic DNA samples were digested, separately, to completionwith four restriction enzymes DraI, EcoRV, HpaI, or PmlI, each of whichgenerates DNA fragments having blunt ends. Double strand adaptors (SEQID NO:9) supplied in the GenomeWalker™ kit were added to the blunt endsof the genomic DNA fragments by DNA ligase. Two rounds of PCR wereperformed to amplify the LTP4 corresponding genomic DNA fragment usingtwo nested primers supplied in the Universal GenomeWalker™ kit that arespecific to the adaptor sequence (AP1 and AP2, for the first and secondadaptor primer, respectively), and two LTP4 gene specific primers (GSP1and GSP2) designed based on the 5′ coding sequence of LTP4 (PSO311306).The oligonucleotide sequences of the four primers are shown in SEQ IDNO:10 (GSP1), SEQ ID NO:11 (AP1), SEQ ID NO:12 (GSP2), and in SEQ IDNO:13 (AP2). The GSP2 primer contains a recognition site for therestriction enzyme NcoI. The AP2 primer from the Universal GenomeWalker™kit contains a SalI restriction site. The 3′ end of the adaptor sequenceSEQ ID NO:9 contains a XmaI recognition site downstream to thecorresponding SalI restriction site in AP2 primer.

The AP1 and the GSP1 primers were used in the first round PCR using eachof the adaptor ligated genomic DNA samples (DraI, EcoRV, HpaI or PmlI)under conditions defined in the GenomeWalker™ protocol. Cycle conditionswere 94° C. for 4 minutes; 35 cycles of 94° C. for 30 seconds, 60° C.for 1 minute, and 68° C. for 3 minutes; and a final 68° C. for 5 minutesbefore holding at 4° C. One microliter from each of the first round PCRproducts was used as templates for the second round PCR with the AP2 andGSP2 primers. Cycle conditions for second round PCR were 94° C. for 4minutes; 25 cycles of 94° C. for 30 seconds, 60° C. for 1 minute, and68° C. for 3 minutes; and a final 68° C. for 5 minutes before holding at4° C. Agarose gels were run to identify specific PCR product with anoptimal fragment length. An approximately 0.6 Kb PCR product wasdetected and subsequently cloned into pCR2.1-TOPO vector by TOPO TAcloning (Invitrogen™). Sequencing of the cloned PCR products revealedthat its 3′ end matched the 5′ end of the PSO311306 cDNA sequence,indicating that the PCR product was indeed the corresponding LTP4genomic DNA fragment. The 508 bp genomic DNA sequence upstream of theputative LTP4 start codon ATG is herein designated as soybean LTP4promoter (SEQ ID NO:1), which includes 8 bp GGGCTGGT non-soybean DNAsequence at the 5′ end derived from the DNA adaptor in the GenomeWalker™kit.

Example 4 LTP4 Promoter Copy Number Analysis

Southern hybridization analysis was performed to determine if there isany other sequence in the soybean genome with high similarity to theLTP4 promoter. Soybean ‘Jack’ wild type genomic DNA was digested withnine different restriction enzymes BamHI, BglII, DraI, EcoRI, EcoRV,HindIII, MfeI, NdeI, and SpeI, each separately, and distributed in a0.7% agarose gel by electrophoresis. The DNA was blotted onto a Nylonmembrane and hybridized in EasyHyb Southern hybridization solution withdigoxigenin (DIG) labeled LTP4 promoter DNA probe, and then sequentiallywashed 10 minutes with 2×SSC/0.1% SDS at room temperature and 3×10minutes at 65° C. with 0.1×SSC/0.1% SDS according to the protocolprovided by the manufacturer (Roche Applied Science, Indianapolis,Ind.). The LTP4 promoter probe was labeled by PCR using the DIG DNAlabeling kit (Roche Applied Science) with primers SEQ ID NO:12 and SEQID NO:13 to make a 539 bp DNA fragment including the entire 508 bp LTP4promoter sequence (SEQ ID NO:1) plus a part of the GenomeWalker™ kit DNAadaptor sequence.

Single band was expected for eight digestions BamHI, BglII, EcoRI,EcoRV, HindIII, MfeI, NdeI, and SpeI if the LTP4 promoter sequence isunique in soybean genome since none of them cut inside the LTP4 probe.As expected, a single band was detected in each of the lanes loaded withDNA digested, respectively, with the above eight restriction enzymes(FIG. 2). Though enzyme DraI would cut LTP4 promoter at position 63 ofLTP4 promoter, the 63 bp sequence would be too short to stably hybridizeto the probe under the stringent Southern hybridization conditions.Single band was also expected for DraI digestion but no band was indeedobserved (FIG. 2). The DraI digestion probably produced a band too smallto be retained on the Southern blot which retained only DNA fragmentslarger than ˜1 Kb. In conclusion, there is only one copy of the LTP4promoter sequence in soybean genome.

Example 5 LTP4:YFP Reporter Constructs and Soybean Transformation

The cloned LTP4 promoter fragment described in EXAMPLE 3 was digestedwith NcoI and XmaI, gel purified using a DNA gel extraction kit (Qiagen,Valencia, Calif.) and cloned into the NcoI and XmaI sites of a Gatewaycloning ready vector QC312 containing the yellow fluorescent reportergene ZS-YELLOW1 N1 (YFP) to make the reporter construct QC372 (SEQ IDNO:25) with the soybean LTP4 promoter driving the YFP gene expression(FIG. 3). The LTP4:YFP expression cassette in construct QC372 was linkedto the SAMS:ALS (S-adenosyl methionine synthetase:acetolactate synthase)expression cassette in construct QC324i (SEQ ID NO:27, FIG. 3) to createconstruct QC384 (SEQ ID NO:26, FIG. 3) by Gateway cloning using LRclonase (Invitrogen™). The linked LTP4:YFP and SAMS:ALS cassettes werereleased as a 5803 bp DNA fragment from construct QC384 by AscIrestriction digestion, separated from the vector backbone fragment byagarose gel electrophoresis, and purified from the gel using a QiagenDNA gel extraction kit. The purified DNA fragment was used to transformsoybean cultivar Jack using the particle gun bombardment method (Kleinet al., Nature 327:70-73 (1987); U.S. Pat. No. 4,945,050) to study theLTP4 promoter activity in stably transformed soybean plants.

Soybean somatic embryos from the Jack cultivar were induced as follows.Cotyledons (smaller than 3 mm in length) were dissected fromsurface-sterilized, immature seeds and were cultured for 6-10 weeksunder fluorescent light at 26° C. on a Murashige and Skoog media (“MSmedia”) containing 0.7% agar and supplemented with 10 mg/ml2,4-dichlorophenoxyacetic acid (2,4-D). Globular stage somatic embryos,which produced secondary embryos, were then excised and placed intoflasks containing liquid MS medium supplemented with 2,4-D (10 mg/ml)and cultured in light on a rotary shaker. After repeated selection forclusters of somatic embryos that multiplied as early, globular stagedembryos, the soybean embryogenic suspension cultures were maintained in35 ml liquid media on a rotary shaker, 150 rpm, at 26° C. withfluorescent lights on a 16:8 hour day/night schedule. Cultures weresubcultured every two weeks by inoculating approximately 35 mg of tissueinto 35 ml of the same fresh liquid MS medium.

Soybean embryogenic suspension cultures were then transformed by themethod of particle gun bombardment using a DuPont Biolistic™ PDS1000/HEinstrument (helium retrofit) (Bio-Rad Laboratories, Hercules, Calif.).To 50 μl of a 60 mg/ml 1.0 mm gold particle suspension were added (inorder): 30 μl of 10 ng/μl LTP4:YFP+SAMS:ALS DNA fragment, 20 μl of 0.1 Mspermidine, and 25 μl of 5 M CaCl₂. The particle preparation was thenagitated for 3 minutes, spun in a centrifuge for 10 seconds and thesupernatant removed. The DNA-coated particles were then washed once in400 μl 100% ethanol and resuspended in 45 μl of 100% ethanol. TheDNA/particle suspension was sonicated three times for one second each. 5μl of the DNA-coated gold particles was then loaded on each macrocarrier disk.

Approximately 300-400 mg of a two-week-old suspension culture was placedin an empty 60×15 mm Petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5 to 10 plates of tissue were bombarded. Membrane rupture pressure wasset at 1100 psi and the chamber was evacuated to a vacuum of 28 inchesmercury. The tissue was placed approximately 3.5 inches away from theretaining screen and bombarded once. Following bombardment, the tissuewas divided in half and placed back into liquid media and cultured asdescribed above.

Five to seven days post bombardment, the liquid media was exchanged withfresh media containing 100 ng/ml chlorsulfuron as selection agent. Thisselective media was refreshed weekly. Seven to eight weeks postbombardment, green, transformed tissue was observed growing fromuntransformed, necrotic embryogenic clusters. Isolated green tissue wasremoved and inoculated into individual flasks to generate new, clonallypropagated, transformed embryogenic suspension cultures. Each clonallypropagated culture was treated as an independent transformation eventand subcultured in the same liquid MS media supplemented with 2,4-D (10mg/ml) and 100 ng/ml chlorsulfuron selection agent to increase mass. Theembryogenic suspension cultures were then transferred to solid agar MSmedia plates without 2,4-D supplement to allow somatic embryos todevelop. A sample of each event was collected at this stage for PCR andquantitative PCR analysis.

Cotyledon stage somatic embryos were dried-down (by transferring theminto an empty small Petri dish that was seated on top of a 10 cm Petridish to allow slow dry down) to mimic the last stages of soybean seeddevelopment. Dried-down embryos were placed on germination solid media,and transgenic soybean plantlets were regenerated. The transgenic plantswere then transferred to soil and maintained in growth chambers for seedproduction.

Genomic DNA was extracted from somatic embryo samples and analyzed byquantitative PCR using the 7500 real time PCR system (AppliedBiosystems) with gene-specific primers and 6-carboxyfluorescein(FAM)-labeled fluorescence probes to check copy numbers of both theSAMS:ALS expression cassette and the LTP4:YFP expression cassette. TheqPCR analysis was done in duplex reactions with a heat shock protein(HSP) gene as the endogenous control and a transgenic DNA sample with aknown single copy of SAMS:ALS or YFP transgene as the calibrator usingthe relative quantification methodology. The endogenous control HSPprobe was labeled with VIC (Applera Corporation, Norwalk, Conn.) and thetarget gene SAMS or YFP probe was labeled with FAM for the simultaneousdetection of both fluorescent probes in the same duplex reactions. Theprimers and probes used in the qPCR analysis are listed below.

SAMS forward primer: SEQ ID NO:16FAM labeled SAMS probe: SEQ ID NO:17SAMS reverse primer: SEQ ID NO:18YFP forward primer: SEQ ID NO:19FAM labeled YFP probe: SEQ ID NO:20YFP reverse primer: SEQ ID NO:21HSP forward primer: SEQ ID NO:22VIC labeled HSP probe: SEQ ID NO:23HSP reverse primer: SEQ ID NO:24FAM labeled DNA oligo probes and VIC labeled oligo probes were obtainedfrom Applied Biosystems while the primers were obtained from MWG-BiotechAG (Bridgeport, Ga.).

Transgenic soybean events containing 1 or 2 copies of both the SAMS:ALSexpression cassette and the LTP4:YFP expression cassette were selectedfor further gene expression evaluation and seed production (see Table2). Events negative for YFP qPCR or with more than 2 copies for the SAMSor YFP qPCR were terminated. YFP expression detection in flowers asdescribed in EXAMPLE 6 is also recorded in the same table.

TABLE 2 Relative transgene copy numbers and YFP expression of LTP4:YFPtransgenic plants Event ID YFP YFP qPCR SAMS qPCR 5138.1.1 + 1.1 1.35138.4.1 + 7.5 5.2 5138.4.3 + 0.8 1.2 5138.4.4 + 0.8 1.1 5138.6.2 + 0.61.3 5138.6.3 + 0.5 0.9 5138.7.3 + 0.7 0.9 5138.7.4 + 0.7 1.0

Example 6 LTP4:YFP Expression in Stable Transgenic Soybean Plants

YFP gene expression was checked at different stages of transgenic plantdevelopment for yellow fluorescence emission under a Leica MZFLIIIstereo microscope equipped with UV light source and appropriate lightfilters (Leica Microsystems Inc., Bannockburn, Ill.). No specific yellowfluorescence was detected during somatic embryo development or invegetative tissues such as leaf, petiole, stem, or root of transgenicplant, or in very young flower bud when flower structure had not formed.Fluorescence was only detected in flower buds and flowers.

A soybean flower consists of five sepals, five petals including onestandard large upper petal, two large side petals, and two small fusedlower petals called kneel to enclose ten stamens and one pistil. Thefilaments of the ten stamens fuse together to form a sheath to enclosethe pistil and separate into 10 branches only at the top to each bear ananther. The pistil consists of a stigma, a style, and an ovary in whichthere are normally 2-4 ovules that will eventually develop into seeds.

No fluorescence was detected in somatic embryos during tissue culture orvegetative tissues such as leaf, root, stem etc. of the LTP4:YFPtransgenic plants. Specific fluorescence signal, white (greyscaledisplay) or bright greenish yellow color (color display) was detectedalmost exclusively in the stigmas of flower bud, open flower, and youngpods (FIG. 4). No fluorescence was detected in the sepals or petals offlower bud (FIG. 4A) or open flower (FIG. 4D). When the flower bud wasopened (FIG. 4B), strong fluorescence was detected in the young stigma.No specific fluorescence was detected in the petals, developing anthers,filaments, style, or the ovary part of the pistil. The dull yellow color(white in grayscale display) in the developing anthers was non-specificsimilar to the dull yellow color from the petals (FIG. 4D). The sameexpression pattern continued to open flower stage though thenon-specific yellow color in anthers became stronger (FIG. 4E). Thestigma-specific fluorescence was better revealed in isolated developingpistil (FIG. 4C) and mature pistil (FIG. 4F). The exposed ovules asindicated by the white arrows (black arrows in grayscale display) didnot show any fluorescence. The stigma remaining on R3 pod carried strongyellow fluorescence (FIG. 4G). The pollen grains attached to the stigmaand style did not emit specific fluorescence. Yellow fluorescence couldstill be detected in the stigma remaining of even older R4 pod (FIG.4H). The stigma remaining of pods older than the R4 pod would be deadand emit auto fluorescence under both YFP and CFP filters.Interestingly, fluorescence was detected in a restricted area of someyoung developing seeds in two of the total eight transgenic events (FIG.4I).

In conclusion, the LTP4:YFP expression was highly specific to thestigmas of developing flowers and young pods. Limited expression inearly developing seeds was also observed but in only two transgenicevents, suggesting that the expression pattern was not universal. Thebiological significance of the highly specialized expression of the LTP4gene in stigma, where pollination involving pollen-stigma interactionsoccurs, still needs to be explored in depth.

1-19. (canceled)
 20. An isolated polynucleotide comprising: (a) anucleotide sequence encoding a polypeptide having lipid proteintransferase activity, wherein the polypeptide has at least 80% sequenceidentity, based on the Clustal method of alignment, when compared to thesequence set forth in SEQ ID NO:15, or (b) a full-length complement ofthe nucleotide sequence of (a).
 21. The isolated polynucleotide of claim20, wherein the polypeptide has at least 85% sequence identity, based onthe Clustal method of alignment, when compared to the sequence set forthin SEQ ID NO:15.
 22. The isolated polynucleotide of claim 20, whereinthe polypeptide has at least 90% sequence identity, based on the Clustalmethod of alignment, when compared to the sequence set forth in SEQ IDNO:15.
 23. The isolated polynucleotide of claim 20, wherein thepolypeptide has at least 95% sequence identity, based on the Clustalmethod of alignment, when compared to the sequence set forth in SEQ IDNO:15.
 24. The isolated polynucleotide of claim 20 encoding the sequenceset forth in SEQ ID NO:15.
 25. The isolated polynucleotide of claim 20,wherein the nucleotide sequence comprises the sequence set forth in SEQID NO:14.
 26. A vector comprising the isolated polynucleotide of claim20.
 27. A recombinant DNA construct comprising the isolatedpolynucleotide of claim 20 operably linked to a regulatory sequence. 28.A cell comprising the recombinant DNA construct of claim
 27. 29. A plantcomprising the recombinant DNA construct of claim
 27. 30. A seedcomprising the recombinant DNA construct of claim
 27. 31. A method fortransforming a cell, comprising transforming a cell with the isolatedpolynucleotide of claim
 20. 32. A method for producing a plantcomprising transforming a plant cell with the isolated polynucleotide ofclaim 20 and regenerating a plant from the transformed plant cell. 33.An isolated polypeptide having lipid protein transferase activity,wherein the isolated polypeptide has at least 80% sequence identity,based on the Clustal method of alignment, when compared to the sequenceset forth in SEQ ID NO:15.
 34. The isolated polypeptide of claim 33,wherein the isolated polypeptide has at least 85% sequence identity,based on the Clustal method of alignment, when compared to the sequenceset forth in SEQ ID NO:15.
 35. The isolated polypeptide of claim 33,wherein the isolated polypeptide has at least 90% sequence identity,based on the Clustal method of alignment, when compared to the sequenceset forth in SEQ ID NO:15.
 36. The isolated polypeptide of claim 33,wherein the isolated polypeptide has at least 95% sequence identity,based on the Clustal method of alignment, when compared to the sequenceset forth in SEQ ID NO:15.
 37. The isolated polypeptide of claim 33,wherein the isolated polypeptide comprises the amino acid sequence setforth in SEQ ID NO:15.